The tryptophan (trp) operon of E. coli consists of 5 contiguous structural genes and their associated control elements. Studies utilizing current techniques in microbial genetics, nucleic acid chemistry, and protein chemistry are proposed in 3 main areas. (1) Structure and function of sites specifying termination of transcription. The nucleotide sequence of 2 termination sites is known, the attenuator (trp a) near the 5' and of the operon, and the terminator (trp t) near the 3' end. Experiments are under way to characterize termination at these sites in a defined in vitro system, with regard to their dependence on RNA polymerase, rho termination factor, the nucleotide sequence, secondary structure, and ionic conditions. Current work suggests that cessation of RNA synthesis is an event separable from dissociation of the termination complex. (2) Ribosome-binding regions and function in a polycistronic mRNA. The relative efficiencies of binding and re-binding of the 6 initiator sites in the trp operon will be investigated. We will attempt to select for mutations permitting "new" initiations of translation, and for mutations altering the characteristics of existing sites, including the one in trp "leader" mRNA of unknown function. The transcript from this region (which is involved in operon regulation in an unknown way) will be tested for its susceptibility to ribonucleases to determine whether "processing" functions are related to its functional significance. (3) Protein-nucleic acid interactions at regulatory sites. Crosslinking studies will be initiated on the interactions of RNA polymerase and rho factor with DNA and its RNA transcript at the two sites of transcription termination in the operon, trp a and trp t, and at the low level non-represssible internal promoter, trp p2. These approaches are expected to yield some understanding of the mechanisms involved in controlling transcription and translation within a bacterial polycistronic operon.